1. Field of the Invention
The present invention relates to molecules inhibiting intercellular adhesion during inflammation and the use of the same. The present invention also relates to using Bst2 protein or fragments thereof as a decoy or Bst2-binding antibody in inhibiting intercellular adhesion and activation of cells participating in inflammation as well as small molecules. The present invention also relates to methods of discovering Bst2 ligand and inhibitor of Bst2 ligand. The present invention is also concerned with a composition comprising the same, and a method for preventing or treating inflammation-associated diseases.
2. General Background and State of the Art
Inflammation is a normal response of the body to protect tissues from infection, injury or diseases. The inflammatory response begins with the production and release of chemical agents by cells in the affected tissues. The chemical agents cause redness, swelling, pain, heat and loss of function. Cells in inflamed tissues generate signals that recruit leukocytes to the site of inflammation. Leukocytes must adhere to endothelial cells to migrate from the bloodstream into the site of inflammation. Also, leukocytes should adhere to antigen-presenting cells to allow normal specific immune responses, and should finally adhere to suitable target cells to lyse pathogen-infected cells, cancer cells, or the like. The recruited leukocytes eliminate any infective or injurious agent and remove debris of damaged cells from the injured tissue.
The infiltrating leukocytes play critical roles in tissue regeneration and immune response in normal inflammation by engulfing invading microorganisms or dead cells. However, the infiltrating leukocytes cause serious or lethal status in pathological chronic inflammation. The abnormal recognition of self cells as non-self (foreign) or excess inflammation by sustained inflammatory responses causes a variety of inflammatory diseases including diabetes mellitus, atherosclerosis, cataract, reperfusion injury, infectious meningitis, rheumatoid arthritis, asthma, sepsis, inflammatory bowel disease and multiple sclerosis.
The interaction between leukocytes and endothelial cells is as follows.
Leukocytes have dual functions to act in a form circulating in the bloodstream or adhering to specific cells. In particular, adherent leukocytes interact with endothelial cells, stabilize intercellular adhesion with antigen-presenting cells or act as effector cells to migrate into inflammatory or infected sites. For normal specific immune response, leukocytes should adhere to antigen-presenting cells and should finally adhere to suitable target cells to lyse pathogen-infected cells, cancer cells, or the like. A massive invasion of leukocytes occurs in an allograft rejection, skin infection or in an injured area, and is also observed in various diseases including degenerative joint diseases, such as osteoarthritis, psoriasis, multiple sclerosis, asthma, rheumatoid arthritis, contact dermatitis and inflammatory bowel disease
In such diseases, greater than 95% of myeloid cells move to and accumulate at the site of inflammation. Leukocytes are crucial agents of the inflammatory response, which exert antimicrobial, secretory and phagocytic activity. They gather in tissues where inflammation is occurring or needs to occur by producing a water-soluble mediator or through specific adhesion to various cells. In fact, anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs (NSAIDs) or glucocorticoid exert therapeutic efficacy by preventing the adhesion and influx of leukocytes. In animal models, the inhibition of intercellular adhesion improves or prevents diseases or allograft rejection in animal models of autoimmune diseases. Recent clinical studies have revealed that humanized monoclonal antibodies inhibiting LFA-1/ICAM-1 or VLA-4/VCAM-1 interaction have significant efficacy and good safety on autoimmue diseases including psoriasis, multiple sclerosis and inflammatory bowel disease.
The uncontrolled invasion of leukocytes into endothelial cells, which is a key feature in the pathogenesis of inflammation-associated diseases, occurs by a multi-step process, which begins with leukocyte adhesion and binding to the surface of endothelial cells. The binding of leukocytes to endothelial cell surface is mediated by cell surface molecules present on the surface of leukocytes and endothelial cells (Bevilacqua, J. Clin. Invest. 11:767-804, 1993). The cell surface molecules are overexpressed as a result of migration of leukocytes from the bloodstream.
The interaction between leukocytes and endothelial cells is a critical factor in many inflammatory diseases. For example, increased leukocyte-endothelial interaction leading to hepatic microperfusion disorders is proposed as a major contributor of hepatic failure (Croner et al., Microvasc. Res. 67:182-191, 2004). For example, atherosclerosis is a typical inflammatory disease in which a number of inflammatory cells including T lymphocytes and activated macrophages are concentrated in the site of atherosclerosis. The accumulation and adhesion of monocytes in discrete segments of arterial endothelium is among the earliest detectable events in atherogenesis and is a central feature of the pathogenesis of atherosclerosis (Ross, Nature 362:801-809, 1993). In this region, proinflammatory cytokines are abundant, which include interferon-gamma and tumor necrosis factor-alpha, regulating regional inflammatory response. A great number of adhesion molecules are expressed on the surface of monocytes (Valente et al., Circulation 86:III20-25, 1992), and endothelial cells overlying atherosclerotic lesions express a number of vascular ligands (Poston et al., Am. J. Pathol, 140:665-673, 1992).
The extravasation of leukocytes across the endothelial barrier is a critical event in the pathogenesis of inflammatory diseases such as rheumatoid arthritis. Endothelial cells participate in the basic mechanism of arthritis, by which various inflammation mediators, such as tumor necrosis factor-alpha and inflammation-inducing cytokines such as interleukin-1 beta, activate endothelial cells. This leads to elevated expression of endothelial cell adhesion molecules in rheumatoid arthritis, resulting in increased interaction between leukocytes and endothelial cells. The recruitment of leukocytes to vascular endothelial cells is also an important step of asthma.
In the airway of patients with asthma, there are increased numbers of activated eosinophils, CD25-positive T lymphocytes and immature macrophages with the phenotypic characteristics of blood monocytes. The expression of HLA class II increases in epithelial cells, macrophages, and other infiltrating cells (Arm et al., Adv. Immunol. 51:323-382, 1992).
An increased rate of leukocyte transmigration across the blood-brain barrier is a major symptom in multiple sclerosis. The interaction between tight junction proteins in leukocytes and those in endothelial cells contributes to the leukocyte extravasation to the central nervous system under physiological conditions, and the altered expression of tight junction proteins is a pathological prerequisite for multiple sclerosis (Worthylake et al., Curr. Opin. Cell Biol. 13:569-577, 2001).
As described above, since the adhesion of leukocytes to endothelial cells is important in a variety of diseases, the inhibition of intercellular adhesion may result in a therapeutic strategy for diverse inflammatory and immune diseases.
With respect to the molecular biology, the following molecules are known to participate in inflammation.
Cytokines: systemic inflammation, which is a general response to serious bacterial infections or traumatic injuries, may affect tissue systems distal to the early damage (Lush and Kvietys, Microcirculation 7:83-101, 2000). Bacterial products and other inflammation-inducing mediators, released from affected tissues, induce the formation of inflammation-inducing mediators including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta, gamma-interferon and interleukin-6. In sepsis, vascular endothelial damage promotes the production of TNF-alpha and interleukin-1 beta. These cytokines directly act on endothelial cells and enhance leukocyte adhesion (Pober et al., J. Immunol. 137:1893-1896, 1986; Dustin and Springer, J. Cell Biol. 107:321-331, 1988; Cotran and Pober, J. Am. Soc. Nephrol. 1:225-235, 1988). These cytokines also activate blood neutrophils in blood and vascular endothelium (Arai et al., Annu Rev Biochem, 59:783-836, 1990). For example, TNF-alpha induces a series of cytokines, chemokines and proteases by an autocrine or paracrine pathway (Ghezzi and Cerami, Methods Mol. Med. 98:1-8. 2004). Interleukin-6 induces mononuclear-endothelial cell interaction and inflammatory damage through expression of adhesion molecules, thus initiating a process of atherosclerosis. Increased blood concentration of interleukin-6 involves vascular inflammation and development of atherosclerosis (Rader, N. Engl. J. Med. 343:1179-1182, 2000). Interleukin-17 induces the expression of many mediators of inflammation, and is involved in the differentiation, maturation and chemotaxis of neutrophil (Witowski et al., Cell Mol Life Sci. 61:567-579, 2004). Increased levels of interleukin-17 have been associated with several pathological conditions, including airway inflammation, rheumatoid arthritis, intraperitoneal abscesses and adhesions, inflammatory bowel disease, allograft rejection, psoriasis, cancer and multiple sclerosis.
Cell surface adhesion molecules: a plurality of inflammatory cytokines induce the expression of endothelial cell-lymphocyte adhesion molecules (ELAMs) on the cell surface (Nortamo et al., Eur. J. Immunol. 21:2629-2632, 1991). They are divided into two classes: intercellular adhesion molecule-1 (ICAM-1) and endothelial cell-lymphocyte adhesion molecule-1 (ELAM-1) (Staunton et al., Cell 52:925-933, 1988). In response to various mediators, vascular endothelium expresses specific cell surface glycoproteins. The binding and extravasation of blood leukocytes are achieved by interaction with a specific ligand or counter receptor (Bevilacqua et al., 1993, 1994). Molecules participating in this process include intercellular adhesion molecule-1 (ICAM-1) as a ligand for CD18, selectins recognizing glycoonjugates on the leukocyte surface, and members of the immunoglobulin superfamily interacting with other members of the same family, leukocyte integrin molecules (Panes et al., J. Physiol. 269:H1955-1964, 1995; Khan et al., Microcirculation 10:351-358, 2003; Nelson et al., Blood 82:3253-3258, 1993; Bevilacqua and Nelson, J. Clin. Invest. 91:379-387, 1993). Leukocyte rolling is regulated by selectins, and transmigration and adhesion of leukocytes on endothelial cells are triggered by the beta 2 integrin, Mac-1 (CD11b/CD18, aMb2, CR3), and LFA-1. Mac-1 and LFA-1 interact with a counter receptor expressed on the surface of endothelial cells, ICAM-1.
Prior art associated with inflammation therapy include the following.
The U.S. Pat. No. 5,367,056 patent describes the inhibition of the binding of polymorphonuclear leukocytes (PMNs) to endothelial cells by treatment of molecules or fragments thereof interrupting the binding to endothelial cell-leukocyte adhesion molecules (ELAMs) as receptors or ligands. This patent also describes antisense nucleotides and ribozymes for suppressing ELAM expression. This patent further describes a method for identifying molecules which inhibit the binding of ELAM to its ligand, and antibodies against ELAM and its ligands.
The U.S. Pat. No. 5,863,540 patent discloses a method of suppressing T cell activation by administering a CD44 protein peptide or a derivative thereof in an amount sufficient to suppress T cell activation. Also disclosed is a method of inhibiting CD44-mediated cell adhesion or CD44-mediated monocyte IL1 release by administering the CD44 protein peptide or derivative thereof in an amount sufficient to inhibit CD44-mediated cell adhesion or monocyte IL1 release. Further disclosed is a method of transporting a drug or cytotoxic agent to a site of inflammation by administering the CD44 protein peptide or derivative thereof linked to the drug or cytotoxic agent.
The U.S. Pat. No. 5,912,266 patent involves the inhibition of intercellular adhesion mediated by the beta 2 integrin family of cell surface molecules. The patent discloses a pharmaceutical composition useful for inhibiting or treating inflammatory and other pathological responses associated with cell adhesion. This patent also discloses a method of inhibiting or treating pathological conditions where leukocytes and lymphocytes cause cellular or tissue damage.
The WO03026692 patent relates to the therapeutic use of an antibody against CD3 antigen complexes in patients with chronic articular inflammation and rheumatoid arthritis.
The EP1304379 patent relates to a humanized anti-CD18 antibody comprising a portion or the whole of an antigen-determining region capable of binding to CD18 antigen.
The U.S. Pat. No. 6,689,869 patent describes the use of a humanized anti-CD18 antibody in inhibiting influx of leukocytes into the lung and other organs during sepsis, and other infectious or non-infectious traumas. The humanized anti-CD18 antibody can be used for inhibiting the ingress of leukocytes into the lung and other organs in patients having endotoxic shock or adult respiratory distress syndrome. The antibody can be administered to treat asthma or leukocyte-mediated reperfusion damage post thrombolytic therapy. Also, the antibody can be used to reduce or eliminate inflammation in a patient being administered with an anti-infective agent, or to assist in the administration of a therapeutic drug to a patient during anticancer chemotherapy.
The U.S. Pat. No. 5,821,336 patent describes polypeptides having a molecular weight of 160 kD, which are mediators or precursors for mediators of inflammation, derivatives thereof, such as mutants and fragments, and processes for their preparation. Nucleotide sequences coding for the polypeptides and derivatives, vectors comprising the nucleotide sequences, antibodies against the polypeptides or their derivatives and antibody derivatives are also disclosed in this patent. Also described are diagnostic and therapeutic methods for inflammatory conditions and Hodgkin's lymphomas using the antibodies and antibody derivatives.
WO2004/039398 discloses a cancer vaccine by generating cytotoxic T cells via utilization of dendritic cells as antigen-presenting cells for the HM1.24 antigen, which is a Bst2 protein. The WO '398 reference also discloses a cancer vaccine containing as an active ingredient an HM1.24 protein or peptide, or a DNA or an RNA encoding an HM1.24 protein or peptide. WO '398 also discloses the use of HM1.24 protein or peptide for cancer vaccine. However, WO '398 fails to disclose or suggest use of soluble HM1.24 protein itself as a therapeutic protein for treating inflammatory diseases such as, but not limited to, atherosclerosis, rheumatoid arthritis, asthma, sepsis, ulcerative colitis, multiple sclerosis, acute myocardial infarction, heart attack, psoriasis, contact dermatitis, osteoarthritis, rhinitis, Crohn's disease and autoimmune diseases. In addition, the mechanistic action of a cancer vaccine is substantially and significantly different from that of a therapeutic protein that inhibits inflammation.
Vaccines, including cancer treatment vaccines, stimulate antigen-specific immune responses directed against the specific protein or protein-containing particles. Protein-containing particles can be specific viruses, bacteria, or infected or otherwise pathogenic cells that express or incorporate the target protein. Inflammation is caused by active or, in the case of pathogenic inflammation, overly active immune cells, and anti-inflammatory therapeutics inhibit inflammation by suppressing the activity of certain immune cells involved in the inflammation.
A cancer vaccine works by activating immune cells to target the specific protein used, while a protein that functions as an anti-inflammatory therapeutic works by inhibiting the activity of immune cells involved in the inflammation. In the case of a cancer vaccine, Bst2 is shown to the immune system as the target that needs to be recognized and eradicated, while in the case of a therapeutic protein, soluble Bst2 protein (Bst2 decoy) itself would actively function to inhibit or suppress the function of certain immune cells. Thus, the use of soluble Bst2 protein as a therapeutic protein for inflammatory diseases is both conceptually and functionally distinct from the use of Bst2 as a cancer vaccine in WO '398.